(VL ¼ κ or λ) chains of individual hmAbs. These VH and VL
amplicons are subsequently incorporated into expression plasmids
using molecular cloning strategies (restriction endonuclease or
ligation-independent) [2]. In vitro production of individual recombinant hmAbs occurs in human embryonic kidney cells (HEK-293)
transfected with cognate VH and VL plasmid pairs; hmAbs secreted
into culture supernatants are subsequently screened for pathogen/
antigen specificity and functional activity.
Low hmAb cloning efficiencies are obtained in studies where
rare antigens are sought, blood sample volume is low or when the
magnitude/timing of the plasmablast component of the immune
response to the pathogen/antigen of interest has not been sufficiently quantified. To counter low hmAb cloning efficiencies,
enrichment for plasmablasts of interest is performed prior to
fluorescence-activated cell sorting (FACS). In vivo enrichment of
human plasmablasts has been achieved by transplantation of irradiated SCID/beige mice with human peripheral blood mononuclear cells (PBMCs) premixed with antigens of interest [3]. In vitro
enrichment has also been achieved by separation of individual cells
into droplets, analyses of the secretome of each cell within the
droplet, and immediate sorting of cells producing the desired antibodies [4]. However, the use of these enrichment methods has not
been widely reported in the literature for a variety of reasons
including the complexity of the techniques and unavailability of
specialist equipment in standard research laboratories.
Recently, a simplified enrichment protocol, known as the
immunoglobulin capture assay (ICA), was described for the
in vitro identification of plasmablasts of interest. In the ICA, a
streptavidin anti-CD45 and biotin anti-human IgG scaffold is
assembled on the surface of plasmablasts to prevent diffusion of
secreted IgG away from the secreting plasmablast. Interactions
between these “captured” IgG molecules and antigens of interest
are subsequently analyzed during FACS. Positive interaction
events, that is, plasmablasts whose IgG have bound to the antigen
of interest, are sorted, while “nonbinders” (non-IgG and nonspecific IgG) are excluded [5]. Production of hmAbs from individual
plasmablasts is subsequently performed as described above. Cloning into expression vectors and expression of hmAbs have been
comprehensively described in published protocols [2, 6].
Only single recombinant vaccine antigens have been utilized in
this assay previously. Here, we demonstrate that a complex probe
such as whole bacterial cells can be efficiently utilized for the
enrichment of pathogen-specific plasmablasts (Fig. 1). Using
formalin-fixed cells representing four capsular variants (6A, 7F,
14, and 19F) of the Gram-positive pneumococcus, we were able
to achieve a hmAb cloning efficiency of ~82%. Our panel of cloned
hmAbs targeted either the 6A, 7F, or 14 capsules—no crossreactivity between structurally dissimilar capsules was discerned.
The modified ICA protocol was also readily applicable to
10
Sara Siris et al.
amplicons are subsequently incorporated into expression plasmids
using molecular cloning strategies (restriction endonuclease or
ligation-independent) [2]. In vitro production of individual recombinant hmAbs occurs in human embryonic kidney cells (HEK-293)
transfected with cognate VH and VL plasmid pairs; hmAbs secreted
into culture supernatants are subsequently screened for pathogen/
antigen specificity and functional activity.
Low hmAb cloning efficiencies are obtained in studies where
rare antigens are sought, blood sample volume is low or when the
magnitude/timing of the plasmablast component of the immune
response to the pathogen/antigen of interest has not been sufficiently quantified. To counter low hmAb cloning efficiencies,
enrichment for plasmablasts of interest is performed prior to
fluorescence-activated cell sorting (FACS). In vivo enrichment of
human plasmablasts has been achieved by transplantation of irradiated SCID/beige mice with human peripheral blood mononuclear cells (PBMCs) premixed with antigens of interest [3]. In vitro
enrichment has also been achieved by separation of individual cells
into droplets, analyses of the secretome of each cell within the
droplet, and immediate sorting of cells producing the desired antibodies [4]. However, the use of these enrichment methods has not
been widely reported in the literature for a variety of reasons
including the complexity of the techniques and unavailability of
specialist equipment in standard research laboratories.
Recently, a simplified enrichment protocol, known as the
immunoglobulin capture assay (ICA), was described for the
in vitro identification of plasmablasts of interest. In the ICA, a
streptavidin anti-CD45 and biotin anti-human IgG scaffold is
assembled on the surface of plasmablasts to prevent diffusion of
secreted IgG away from the secreting plasmablast. Interactions
between these “captured” IgG molecules and antigens of interest
are subsequently analyzed during FACS. Positive interaction
events, that is, plasmablasts whose IgG have bound to the antigen
of interest, are sorted, while “nonbinders” (non-IgG and nonspecific IgG) are excluded [5]. Production of hmAbs from individual
plasmablasts is subsequently performed as described above. Cloning into expression vectors and expression of hmAbs have been
comprehensively described in published protocols [2, 6].
Only single recombinant vaccine antigens have been utilized in
this assay previously. Here, we demonstrate that a complex probe
such as whole bacterial cells can be efficiently utilized for the
enrichment of pathogen-specific plasmablasts (Fig. 1). Using
formalin-fixed cells representing four capsular variants (6A, 7F,
14, and 19F) of the Gram-positive pneumococcus, we were able
to achieve a hmAb cloning efficiency of ~82%. Our panel of cloned
hmAbs targeted either the 6A, 7F, or 14 capsules—no crossreactivity between structurally dissimilar capsules was discerned.
The modified ICA protocol was also readily applicable to
10
Sara Siris et al.